UDP-Glucose/P2Y14 Receptor Signaling Exacerbates Neuronal Apoptosis After Subarachnoid Hemorrhage in Rats.

BACKGROUND: Subarachnoid hemorrhage (SAH) is a severe subtype of stroke with poor outcomes. Abnormal glucose metabolism often occurs after SAH, but the strict control of blood glucose levels is not always beneficial. This study aimed to investigate the contribution of uridine diphosphate glucose (UDP-G), an intermediate of glucose/glycogen metabolism, and its receptor P2Y14 (P2Y purinoceptor 14) to SAH pathology and explored the potential targeted treatments in rats. METHODS: A total of 218 Sprague-Dawley male rats were used. SAH was induced by endovascular perforation. Brain expressions of P2Y14, uridine diphosphate glucose (UDP-G), and its converting enzyme UGP2 (UDP-G pyrophosphorylase-2) were evaluated. Exogenous UDP-G or selective P2Y14 inhibitor was administered intranasally at 1 hour after SAH to explore their potential effects. Intranasal Ugp2 or P2ry14 siRNA was delivered 24 hours before SAH for mechanistic evaluation. Primary neuron culture and hemoglobin stimulation were used as in vitro model of SAH. Post-SAH evaluation included liquid chromatography-mass spectrometry measurement of brain endogenous UDP-G level, neurobehavioral assessments, Western blotting, immunohistochemistry, TUNEL staining, and Nissl staining. RESULTS: There was an acute elevation of endogenous brain UDP-G and UGP2 after SAH, and P2Y14 was expressed in neurons. Although P2Y14 inhibitor decreased neurological dysfunction, neuronal apoptosis, and proapoptotic molecules, exogenous UDP-G exacerbated these outcomes at 24 hours after SAH. Early inhibition of P2Y14 preserved long-term neuronal survival in the hippocampus, amygdala, and cortex with improved neurocognition and depressive-like behavior. In addition, in vivo knockdown of Ugp2- and P2ry14-reduced neurological deficits and proapoptotic molecules at 24 hours after SAH, and furthermore in vitro knockdown of P2ry14-reduced apoptosis in hemoglobin stimulated primary neuron. CONCLUSIONS: These findings suggest a detrimental role of brain UDP-G/P2Y14 signaling in SAH, as a part of glucose metabolic pathology at the tissue level. P2Y14 inhibitor 4-[4-(4-piperidinyl)phenyl]-7-[4-(trifluoromethyl)phenyl]-2-naphthalenecarboxylic acid hydrochloride may serve as a potential therapeutic target in treating patients with SAH.

Photocatalytic Nitrogen Fixation Coupled with the Generation of Value-Added Chemicals from N2 and Cellulose over MoO3 Nanosheets.

Photocatalytic nitrogen fixation from N2 provides an alternative strategy for ammonia (NH3) production, but it was limited by the consumption of a sacrificial electron donor for the currently reported half-reaction system. Here, we use naturally abundant and renewable cellulose as the sacrificial reagent for photocatalytic nitrogen fixation over oxygen-vacancy-modified MoO3 nanosheets as the photocatalyst. In this smartly designed photocatalytic system, the photooxidation of cellulose not only generates value-added chemicals but also provides electrons for the N2 reduction reaction and results in the production of NH3 with a maximum rate of 68 µmol·h-1·g-1. Also, the oxygen vacancies provide efficient active sites for both cellulose oxygenolysis and nitrogen fixation reactions. This work represents useful inspiration for realizing nitrogen fixation coupled with the generation of value-added chemicals from N2 and cellulose through a photocatalysis strategy.

Accelerated Charge Transfer through Interface Chemical Bonds in MoS2/TiO2 for Photocatalytic Conversion of Lignocellulosic Biomass to H2.

Solar photocatalytic H2 production from lignocellulosic biomass has attracted great interest, but it suffers from low photocatalytic efficiency owing to the absence of highly efficient photocatalysts. Herein, we designed and constructed ultrathin MoS2-modified porous TiO2 microspheres (MT) with abundant interface Ti-S bonds as photocatalysts for photocatalytic H2 generation from lignocellulosic biomass. Owing to the accelerated charge transfer related to Ti-S bonds, as well as the abundant active sites for both H2 and ●OH generation, respectively, related to the high exposed edge of MoS2 and the large specific surface area of TiO2, MT photocatalysts demonstrate good performance in the photocatalytic conversion of α-cellulose and lignocellulosic biomass to H2. The highest H2 generation rate of 849 µmol·g-1·h-1 and apparent quantum yield of 4.45% at 380 nm was achieved in α-cellulose aqueous solution for the optimized MT photocatalyst. More importantly, lignocellulosic biomass of corncob, rice hull, bamboo, polar wood chip, and wheat straw were successfully converted to H2 over MT photocatalysts with H2 generation rate of 10, 19, 36, 29, and 8 µmol·g-1·h-1, respectively. This work provides a guiding design approach to develop highly active photocatalysts via interface engineering for solar H2 production from lignocellulosic biomass.

Fractalkine Enhances Hematoma Resolution and Improves Neurological Function via CX3CR1/AMPK/PPARγ Pathway After GMH.

BACKGROUND: Hematoma clearance has been a proposed therapeutic strategy for hemorrhagic stroke. This study investigated the impact of CX3CR1 (CX3C chemokine receptor 1) activation mediated by r-FKN (recombinant fractalkine) on hematoma resolution, neuroinflammation, and the underlying mechanisms involving AMPK (AMP-activated protein kinase)/PPARγ (peroxisome proliferator-activated receptor gamma) pathway after experimental germinal matrix hemorrhage (GMH). METHODS: A total of 313 postnatal day 7 Sprague Dawley rat pups were used. GMH was induced using bacterial collagenase by a stereotactically guided infusion. r-FKN was administered intranasally at 1, 25, and 49 hours after GMH for short-term neurological evaluation. Long-term neurobehavioral tests (water maze, rotarod, and foot-fault test) were performed 24 to 28 days after GMH with the treatment of r-FKN once daily for 7 days. To elucidate the underlying mechanism, CX3CR1 CRISPR, or selective CX3CR1 inhibitor AZD8797, was administered intracerebroventricularly 24 hours preinduction of GMH. Selective inhibition of AMPK/PPARγ signaling in microglia via intracerebroventricularly delivery of liposome-encapsulated specific AMPK (Lipo-Dorsomorphin), PPARγ (Lipo-GW9662) inhibitor. Western blot, Immunofluorescence staining, Nissl staining, Hemoglobin assay, and ELISA assay were performed. RESULTS: The brain expression of FKN and CX3CR1 were elevated after GMH. FKN was expressed on both neurons and microglia, whereas CX3CR1 was mainly expressed on microglia after GMH. Intranasal administration of r-FKN improved the short- and long-term neurobehavioral deficits and promoted M2 microglia polarization, thereby attenuating neuroinflammation and enhancing hematoma clearance, which was accompanied by an increased ratio of p-AMPK (phosphorylation of AMPK)/AMPK, Nrf2 (nuclear factor erythroid 2-related factor 2), PPARγ, CD36 (cluster of differentiation 36), CD163 (hemoglobin scavenger receptor), CD206 (the mannose receptor), and IL (interleukin)-10 expression, and decreased CD68 (cluster of differentiation 68), IL-1ß, and TNF (tumor necrosis factor) α expression. The administration of CX3CR1 CRISPR or CX3CR1 inhibitor (AZD8797) abolished the protective effect of FKN. Furthermore, selective inhibition of microglial AMPK/PPARγ signaling abrogated the anti-inflammation effects of r-FKN after GMH. CONCLUSIONS: CX3CR1 activation by r-FKN promoted hematoma resolution, attenuated neuroinflammation, and neurological deficits partially through the AMPK/PPARγ signaling pathway, which promoted M1/M2 microglial polarization. Activating CX3CR1 by r-FKN may provide a promising therapeutic approach for treating patients with GMH.

Aprepitant attenuates NLRC4-dependent neuronal pyroptosis via NK1R/PKCδ pathway in a mouse model of intracerebral hemorrhage.

BACKGROUND: Pyroptosis is a programmed cell death mediated by inflammasomes. Previous studies have reported that inhibition of neurokinin receptor 1 (NK1R) exerted neuroprotection in several neurological diseases. Herein, we have investigated the role of NK1R receptor inhibition using Aprepitant to attenuate NLRC4-dependent neuronal pyroptosis after intracerebral hemorrhage (ICH), as well as the underlying mechanism. METHODS: A total of 182 CD-1 mice were used. ICH was induced by injection of autologous blood into the right basal ganglia. Aprepitant, a selective antagonist of NK1R, was injected intraperitoneally at 1 h after ICH. To explore the underlying mechanism, NK1R agonist, GR73632, and protein kinase C delta (PKCδ) agonist, phorbol 12-myristate 13-acetate (PMA), were injected intracerebroventricularly at 1 h after ICH induction, and small interfering ribonucleic acid (siRNA) for NLRC4 was administered via intracerebroventricular injection at 48 h before ICH induction, respectively. Neurobehavioral tests, western blot, and immunofluorescence staining were performed. RESULTS: The expression of endogenous NK1R and NLRC 4 were gradually increased after ICH. NK1R was expressed on neurons. Aprepitant significantly improved the short- and long-term neurobehavioral deficits after ICH, which was accompanied with decreased neuronal pyroptosis, as well as decreased expression of NLRC4, Cleaved-caspase-1, GSDMD (gasdermin D), IL-1ß, and IL-18. Activation of NK1R or PKCδ abolished these neuroprotective effects of Aprepitant after ICH. Similarly, knocking down NLRC4 using siRNA produced similar neuroprotective effects. CONCLUSION: Aprepitant suppressed NLRC4-dependent neuronal pyroptosis and improved neurological function, possibly mediated by inhibition of NK1R/PKCδ signaling pathways after ICH. The NK1R may be a promising therapeutic target for the treatment of ICH.

CCR5 Activation Promotes NLRP1-Dependent Neuronal Pyroptosis via CCR5/PKA/CREB Pathway After Intracerebral Hemorrhage.

BACKGROUND AND PURPOSE: Neuronal pyroptosis is a type of regulated cell death triggered by proinflammatory signals. CCR5 (C-C chemokine receptor 5)-mediated inflammation is involved in the pathology of various neurological diseases. This study investigated the impact of CCR5 activation on neuronal pyroptosis and the underlying mechanism involving cAMP-dependent PKA (protein kinase A)/CREB (cAMP response element binding)/NLRP1 (nucleotide-binding domain leucine-rich repeat pyrin domain containing 1) pathway after experimental intracerebral hemorrhage (ICH). METHODS: A total of 194 adult male CD1 mice were used. ICH was induced by autologous whole blood injection. Maraviroc (MVC)-a selective antagonist of CCR5-was administered intranasally 1 hour after ICH. To elucidate the underlying mechanism, a specific CREB inhibitor, 666-15, was administered intracerebroventricularly before MVC administration in ICH mice. In a set of naive mice, rCCL5 (recombinant chemokine ligand 5) and selective PKA activator, 8-Bromo-cAMP, were administered intracerebroventricularly. Short- and long-term neurobehavioral assessments, Western blot, Fluoro-Jade C, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and immunofluorescence staining were performed. RESULTS: The brain expression of CCL5 (chemokine ligand 5), CCR5, PKA-Cα (protein kinase A-Cα), p-CREB (phospho-cAMP response element binding), and NLRP1 was increased, peaking at 24 hours after ICH. CCR5 was expressed on neurons, microglia, and astrocytes. MVC improved the short- and long-term neurobehavioral deficits and decreased neuronal pyroptosis in ipsilateral brain tissues at 24 hours after ICH, which were accompanied by increased PKA-Cα and p-CREB expression, and decreased expression of NLRP1, ASC (apoptosis-associated speck-like protein containing a CARD), C-caspase-1, GSDMD (gasdermin D), and IL (interleukin)-1ß/IL-18. Such effects of MVC were abolished by 666-15. At 24 hours after injection in naive mice, rCCL5 induced neurological deficits, decreased PKA-Cα and p-CREB expression in the brain, and upregulated NLRP1, ASC, C-caspase-1, N-GSDMD, and IL-1ß/IL-18 expression. Those effects of rCCL5 were reversed by 8-Bromo-cAMP. CONCLUSIONS: CCR5 activation promoted neuronal pyroptosis and neurological deficits after ICH in mice, partially through the CCR5/PKA/CREB/NLRP1 signaling pathway. CCR5 inhibition with MVC may provide a promising therapeutic approach in managing patients with ICH.

TREM (Triggering Receptor Expressed on Myeloid Cells)-1 Inhibition Attenuates Neuroinflammation via PKC (Protein Kinase C) δ/CARD9 (Caspase Recruitment Domain Family Member 9) Signaling Pathway After Intracerebral Hemorrhage in Mice.

Background and Purpose: Intracerebral hemorrhage (ICH) is a devastating subtype of stroke with high mortality and disability. Inflammatory response promotes secondary brain injury after ICH. TREM (triggering receptor expressed on myeloid cells)-1 is a key regulator of inflammation. The aim of this study was to evaluate the role of TREM-1 in neuroinflammatory response after ICH in mice. Methods: CD1 mice (n=275) were used in this study. Mice were subjected to ICH by autologous blood injection. TREM-1 knockout CRISPR was administered intracerebroventricularly to evaluate the role of TREM-1 after ICH. A selective TREM-1 inhibitor, LP17, was administered intranasally 2 hours after ICH. To elucidate TREM-1 signaling pathway, CARD9 (caspase recruitment domain family member 9) activation CRISPR was administered with LP17 and TREM-1 activating anti-mouse TREM-1 monoclonal antibody (mAb) was administered with Rottlerin, a specific PKC (protein kinase C) δ inhibitor. Lastly, to evaluate the role of HMGB1 (high-mobility group box 1) in TREM-1 mediated microglia activation, glycyrrhizin, an inhibitor of HMBG1 was administered with TREM-1 activating mAb. Neurobehavioral test, brain water content, Western blot, immunofluorescence staining, and coimmunoprecipitation was performed. Results: TREM-1 knockout reduced ICH-induced neurobehavioral deficits and neuroinflammatory response. The temporal expression of HMGB1, TREM-1, PKC δ, and CARD9 increased after ICH. TREM-1 was expressed on microglia. Intranasal administration of LP17 significantly decreased brain edema and improved neurobehavioral outcomes at 24 and 72 hours after ICH. LP17 promoted M2 microglia polarization and reduced proinflammatory cytokines after ICH, which was reversed with CARD9 activation CRISPR. TREM-1 mAb increased neurobehavior deficits, proinflammatory cytokines, and reduced M2 microglia after ICH, which was reversed with Rottlerin. HMBG1 interaction with TREM-1 increased after ICH, and glycyrrhizin reduced neuroinflammation and promoted M2 microglia which was reversed with TREM-1 mAb. Conclusions: This study demonstrated that TREM-1 enhanced neuroinflammation by modulating microglia polarization after ICH, and this regulation was partly mediated via PKC δ/CARD9 signaling pathway and increased HMGB1 activation of TREM-1.

TGR5 activation attenuates neuroinflammation via Pellino3 inhibition of caspase-8/NLRP3 after middle cerebral artery occlusion in rats.

BACKGROUND: Nucleotide-binding oligomerization domain-like receptor pyrin domain-containing protein 3 (NLRP3) plays an important role in mediating inflammatory responses during ischemic stroke. Bile acid receptor Takeda-G-protein-receptor-5 (TGR5) has been identified as an important component in regulating brain inflammatory responses. In this study, we investigated the mechanism of TGR5 in alleviating neuroinflammation after middle cerebral artery occlusion (MCAO). METHODS: Sprague-Dawley rats were subjected to MCAO and TGR5 agonist INT777 was administered intranasally 1 h after MCAO. Small interfering RNAs (siRNA) targeting TGR5 and Pellino3 were administered through intracerebroventricular injection 48 h before MCAO. Infarct volumes and neurologic scores were evaluated, and ELISA, flow cytometry, immunofluorescence staining, immunoblotting, and co-immunoprecipitation were used for the evaluations. RESULTS: Endogenous TGR5 and Pellino3 levels increased after MCAO. TGR5 activation by INT777 significantly decreased pro-inflammatory cytokine, cleaved caspase-8, and NLRP3 levels, thereby reducing brain infarctions; both short- and long-term neurobehavioral assessments showed improvements. Ischemic damage induced the interaction of TGR5 with Pellino3. Knockdown of either TGR5 or Pellino3 increased the accumulation of cleaved caspase-8 and NLRP3, aggravated cerebral impairments, and abolished the anti-inflammatory effects of INT777 after MCAO. CONCLUSIONS: TGR5 activation attenuated brain injury by inhibiting neuroinflammation after MCAO, which could be mediated by Pellino3 inhibition of caspase-8/NLRP3.

Recombinant CCL17-dependent CCR4 activation alleviates neuroinflammation and neuronal apoptosis through the PI3K/AKT/Foxo1 signaling pathway after ICH in mice.

BACKGROUND: Intracerebral hemorrhage (ICH), a devastating subtype of stroke, is associated with high mortality and morbidity. Neuroinflammation is an important factor leading to ICH-induced neurological injuries. C-C Chemokine Receptor 4 (CCR4) plays an important role in enhancing hematoma clearance after ICH. However, it is unclear whether CCR4 activation can ameliorate neuroinflammation and apoptosis of neurons following ICH. The aim of the present study was to examine the effects of recombinant CCL17 (rCCL17)-dependent CCR4 activation on neuroinflammation and neuronal apoptosis in an intrastriatal autologous blood injection ICH model, and to determine whether the PI3K/AKT/Foxo1 signaling pathway was involved. METHODS: Two hundred twenty-six adult (8-week-old) male CD1 mice were randomly assigned to sham and ICH surgery groups. An intrastriatal autologous blood injection ICH model was used. rCCL17, a CCR4 ligand, was delivered by intranasal administration at 1 h, 3 h, and 6 h post-ICH. CCL17 antibody was administrated by intraventricular injection at 1 h post-ICH. C021, a specific inhibitor of CCR4 and GDC0068, an AKT inhibitor were delivered intraperitoneally 1 h prior to ICH induction. Brain edema, neurobehavioral assessments, western blotting, Fluoro-Jade C staining, terminal deoxynucleotidyl transferase dUTP nick end labeling, and immunofluorescence staining were conducted. RESULTS: Endogenous expression of CCL17 and CCR4 were increased following ICH, peaking at 5 days post-induction. CCR4 was found to co-localize with microglia, neurons, and astrocytes. rCCL17 treatment decreased brain water content, attenuated short- and long-term neurological deficits, deceased activation of microglia/macrophages and infiltration of neutrophils, and inhibited neuronal apoptosis in the perihematomal region post-ICH. Moreover, rCCL17 treatment post-ICH significantly increased the expression of CCR4, PI3K, phosphorylated AKT, and Bcl-2, while Foxo1, IL-1ß, TNF-α, and Bax expression were decreased. The neuroprotective effects of rCCL17 were reversed with the administration of C021 or GDC0068. CONCLUSIONS: rCCL17-dependent CCR4 activation ameliorated neurological deficits, reduced brain edema, and ameliorated neuroinflammation and neuronal apoptosis, at least in part, through the PI3K/AKT/Foxo1 signaling pathway after ICH. Thus, activation of CCR4 may provide a promising therapeutic approach for the early management of ICH.

Activation of GPR39 with TC-G 1008 attenuates neuroinflammation via SIRT1/PGC-1α/Nrf2 pathway post-neonatal hypoxic-ischemic injury in rats.

BACKGROUND: Hypoxic-ischemic encephalopathy (HIE) is a severe anoxic brain injury that leads to premature mortality or long-term disabilities in infants. Neuroinflammation is a vital contributor to the pathogenic cascade post-HIE and a mediator to secondary neuronal death. As a plasma membrane G-protein-coupled receptor, GPR39, exhibits anti-inflammatory activity in several diseases. This study aimed to explore the neuroprotective function of GPR39 through inhibition of inflammation post-hypoxic-ischemic (HI) injury and to elaborate the contribution of sirtuin 1(SIRT1)/peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α)/nuclear factor, erythroid 2 like 2(Nrf2) in G-protein-coupled receptor 39 (GPR39)-mediated protection. METHODS: A total of 206 10-day-old Sprague Dawley rat pups were subjected to HIE or sham surgery. TC-G 1008 was administered intranasally at 1 h, 25 h, 49 h, and 73 h post-HIE induction. SIRT1 inhibitor EX527, GPR39 CRISPR, and PGC-1α CRISPR were administered to elucidate the underlying mechanisms. Brain infarct area, short-term and long-term neurobehavioral tests, Nissl staining, western blot, and immunofluorescence staining were performed post-HIE. RESULTS: The expression of GPR39 and pathway-related proteins, SIRT1, PGC-1α and Nrf2 were increased in a time-dependent manner, peaking at 24 h or 48-h post-HIE. Intranasal administration of TC-G 1008 reduced the percent infarcted area and improved short-term and long-term neurological deficits. Moreover, TC-G 1008 treatment significantly increased the expression of SIRT1, PGC-1α and Nrf2, but downregulated the expressions of IL-6, IL-1ß, and TNF-α. GPR39 CRISPR EX527 and PGC-1α CRISPR abolished GPR39's neuroprotective effects post-HIE. CONCLUSIONS: TC-G 1008 attenuated neuroinflammation in part via the SIRT1/PGC-1α/Nrf2 pathway in a neonatal rat model of HIE. TC-G 1008 may be a novel therapeutic target for treatment post-neonatal HIE injury.

Activation of MC1R with BMS-470539 attenuates neuroinflammation via cAMP/PKA/Nurr1 pathway after neonatal hypoxic-ischemic brain injury in rats.

BACKGROUND: Microglia-mediated neuroinflammation plays a crucial role in the pathogenesis of hypoxic-ischemic (HI)-induced brain injury. Activation of melanocortin-1 receptor (MC1R) has been shown to exert anti-inflammatory and neuroprotective effects in several neurological diseases. In the present study, we have explored the role of MC1R activation on neuroinflammation and the potential underlying mechanisms after neonatal hypoxic-ischemic brain injury in rats. METHODS: A total of 169 post-natal day 10 unsexed rat pups were used. HI was induced by right common carotid artery ligation followed by 2.5 h of hypoxia. BMS-470539, a specific selective MC1R agonist, was administered intranasally at 1 h after HI induction. To elucidate the potential underlying mechanism, MC1R CRISPR KO plasmid or Nurr1 CRISPR KO plasmid was administered via intracerebroventricular injection at 48 h before HI induction. Percent brain infarct area, short- and long-term neurobehavioral tests, Nissl staining, immunofluorescence staining, and Western blot were conducted. RESULTS: The expression levels of MC1R and Nurr1 increased over time post-HI. MC1R and Nurr1 were expressed on microglia at 48 h post-HI. Activation of MC1R with BMS-470539 significantly reduced the percent infarct area, brain atrophy, and inflammation, and improved short- and long-term neurological deficits at 48 h and 28 days post-HI. MC1R activation increased the expression of CD206 (a microglial M2 marker) and reduced the expression of MPO. Moreover, activation of MC1R with BMS-470539 significantly increased the expression levels of MC1R, cAMP, p-PKA, and Nurr1, while downregulating the expression of pro-inflammatory cytokines (TNFα, IL-6, and IL-1ß) at 48 h post-HI. However, knockout of MC1R or Nurr1 by specific CRISPR reversed the neuroprotective effects of MC1R activation post-HI. CONCLUSIONS: Our study demonstrated that activation of MC1R with BMS-470539 attenuated neuroinflammation, and improved neurological deficits after neonatal hypoxic-ischemic brain injury in rats. Such anti-inflammatory and neuroprotective effects were mediated, at least in part, via the cAMP/PKA/Nurr1 signaling pathway. Therefore, MC1R activation might be a promising therapeutic target for infants with hypoxic-ischemic encephalopathy (HIE).

Activation of GPR40 attenuates neuroinflammation and improves neurological function via PAK4/CREB/KDM6B pathway in an experimental GMH rat model.

BACKGROUND: Germinal matrix hemorrhage (GMH) is defined by the rupture of immature blood vessels in the germinal matrix, where subsequent hemorrhage enters the subependymal zone and the cerebral lateral ventricles. The consequent blood clot has been identified as the causative factor of secondary brain injury, which triggers a series of complex parallel and sequential harmful mechanisms, including neuroinflammation. The orphan G-protein-coupled receptor 40 (GPR40), a free fatty acid (FFA) receptor 1, has been shown to exert anti-inflammatory effects when activated and improved outcomes in animal models of stroke. We aimed to investigate the anti-inflammatory effects of GPR40 and its underlying mechanisms after GMH. METHODS: GMH model was induced in 7-day-old rat pups by an intraparenchymal injection of bacterial collagenase. GPR40 agonist, GW9508, was administered intranasally 1 h, 25 h, and 49 h after GMH induction. CRISPR targeting GPR40, PAK4, and KDM6B were administered through intracerebroventricular injection 48 h before GMH induction. Neurologic scores, microglia polarization, and brain morphology were evaluated by negative geotaxis, right reflex, rotarod test, foot fault test, Morris water maze, immunofluorescence staining, Western blots, and nissl staining respectfully. RESULTS: The results demonstrated that GW9508 improved neurological and morphological outcomes after GMH in the short (24 h, 48 h, 72h) and long-term (days 21-27). However, the neuroprotective effects of treatment were abolished by GW1100, a selective GPR40 antagonist. GW9508 treatment increased populations of M2 microglia and decreased M1 microglia in periventricular areas 24 h after GMH induction. GW9508 upregulated the phosphorylation of PAK4, CREB, and protein level of KDM6B, CD206, IL-10, which was also met with the downregulation of inflammatory markers IL-1ß and TNF-α. The mechanism study demonstrated that the knockdown of GPR40, PAK4, and KDM6B reversed the neuroprotective effects brought on by GW9508. This evidence suggests that GPR40/PAK4/CREB/KDM6B signaling pathway in microglia plays a role in the attenuation of neuroinflammation after GMH. CONCLUSIONS: In conclusion, the present study demonstrates that the activation of GPR40 attenuated GMH-induced neuroinflammation through the activation of the PAK4/CREB/KDM6B signaling pathway, and M2 microglia may be a major mediator of this effect. Thus, GPR40 may serve as a potential target in the reduction of the inflammatory response following GMH, thereby improving neurological outcomes in the short- and long-term.

INT-777 attenuates NLRP3-ASC inflammasome-mediated neuroinflammation via TGR5/cAMP/PKA signaling pathway after subarachnoid hemorrhage in rats.

BACKGROUND: Inflammasome-mediated neuroinflammation plays an important role in the pathogenesis of early brain injury (EBI) following subarachnoid hemorrhage (SAH). The activation of the TGR5 receptor has been shown to be neuroprotective in a variety of neurological diseases. This study aimed to investigate the effects of the specific synthetic TGR5 agonist, INT-777, in attenuating NLRP3-ASC inflammasome activation and reducing neuroinflammation after SAH. METHODS: One hundred and eighty-four male Sprague Dawley rats were used. SAH was induced by the endovascular perforation. INT-777 was administered intranasally at 1 h after SAH induction. To elucidate the signaling pathway involved in the effect of INT-777 on inflammasome activation during EBI, TGR5 knockout CRISPR and PKA inhibitor H89 were administered intracerebroventricularly and intraperitoneally at 48 h and 1 h before SAH. The SAH grade, short- and long-term neurobehavioral assessments, brain water content, western blot, immunofluorescence staining, and Nissl staining were performed. RESULTS: The expressions of endogenous TGR5, p-PKA, and NLRP3-ASC inflammasome were increased after SAH. INT-777 administration significantly decreased NLRP3-ASC inflammasome activation in microglia, reduced brain edema and neuroinflammation, leading to improved short-term neurobehavioral functions at 24 h after SAH. The administration of TGR5 CRISPR or PKA inhibitor (H89) abolished the anti-inflammation effects of INT-777, on NLRP3-ASC inflammasome, pro-inflammatory cytokines (IL-6, IL-1ß, and TNF-a), and neutrophil infiltration at 24 h after SAH. Moreover, early administration of INT-777 attenuated neuronal degeneration in hippocampus on 28 d after SAH. CONCLUSIONS: INT-777 attenuated NLRP3-ASC inflammasome-dependent neuroinflammation in the EBI after SAH, partially via TGR5/cAMP/PKA signaling pathway. Early administration of INT-777 may serve as a potential therapeutic strategy for EBI management in the setting of SAH.

Inhibition of lysophosphatidic acid receptor 1 attenuates neuroinflammation via PGE2/EP2/NOX2 signalling and improves the outcome of intracerebral haemorrhage in mice.

Lysophosphatidic acid receptor 1 (LPA1) plays a critical role in proinflammatory processes in the central nervous system by modulating microglia activation. The aim of this study was to explore the anti-inflammatory effects and neurological function improvement of LPA1 inhibition after intracerebral haemorrhage (ICH) in mice and to determine whether prostaglandin E2 (PGE2), E-type prostaglandin receptor 2 (EP2), and NADPH oxidase 2 (NOX2) signalling are involved in LPA1-mediated neuroinflammation. ICH was induced in CD1 mice by autologous whole blood injection. AM966, a selective LPA1 antagonist, was administered by oral gavage 1 h and 12 h after ICH. The LPA1 endogenous ligand, LPA was administered to verify the effect of LPA1 activation. To elucidate potential inflammatory mechanisms of LPA1, the selective EP2 activator butaprost was administered by intracerebroventricular injection with either AM966 or LPA1 CRISPR knockout (KO). Water content of the brain, neurobehavior, immunofluorescence staining, and western blot were performed. After ICH, EP2 was expressed in microglia whereas LPA1 was expressed in microglia, neurons, and astrocytes, which peaked after 24 h. AM966 inhibition of LPA1 improved neurologic function, reduced brain oedema, and suppressed perihematomal inflammatory cells after ICH. LPA administration aggravated neurological deficits after ICH. AM966 treatment and LPA1 CRISPR KO both decreased the expressions of PGE2, EP2, NOX2, NF-κB, TNF-α, IL-6, and IL-1ß expressions after ICH, which was reversed by butaprost. This study demonstrated that inhibition of LPA1 attenuated neuroinflammation caused by ICH via PGE2/EP2/NOX2 signalling pathway in mice, which consequently improved neurobehavioral functions and alleviated brain oedema. LPA1 may be a promising therapeutic target to attenuate ICH-induced secondary brain injury.

GW0742 activates miR-17-5p and inhibits TXNIP/NLRP3-mediated inflammation after hypoxic-ischaemic injury in rats and in PC12 cells.

This study aimed to investigate the effects of PPAR-ß/δ receptor agonist GW0742 on neuroinflammation in a rat model of hypoxia-ischaemia (HI) and in PC12 cells in OGD model. HI was induced by ligating the common carotid artery and inducing hypoxia for 150 minutes. Immunofluorescence was used for quantification of microglia activation and for determining cellular localization of PPAR-ß/δ. Expression of proteins was measured by Western blot. Activation of miR-17-5p by GW0742 was assessed in PC12 cells by Dual-Luciferase Reporter Gene Assay. The endogenous expression of TXNIP, NLRP3, cleaved caspase-1 and IL-1ß was increased after HI. GW0742 treatment significantly reduced the number of activated pro-inflammatory microglia in ipsilateral hemisphere after HI. Mechanistically, GW0742 significantly decreased the expression of TXNIP, NLRP3, IL-6 and TNF-α. Either PPAR-ß/δ antagonist GSK3787, miR-17-5p inhibitor, or TXNIP CRISPR activation abolished the anti-inflammatory effects of GW0742. Activation of PPAR-ß/δ by GW0742 activated miR-17-5p expression in PC12 cells and increased cell viability after OGD, which was accompanied by decreased expression of TXNIP and reduced secretion of IL-1ß and TNF-α. In conclusion, GW0742 may be a promising neurotherapeutic for the management of HI patients.

Inhibition of EZH2 (Enhancer of Zeste Homolog 2) Attenuates Neuroinflammation via H3k27me3/SOCS3/TRAF6/NF-κB (Trimethylation of Histone 3 Lysine 27/Suppressor of Cytokine Signaling 3/Tumor Necrosis Factor Receptor Family 6/Nuclear Factor-κB) in a Rat Model of Subarachnoid Hemorrhage.

BACKGROUND AND PURPOSE: Neuroinflammation has been proven to play an important role in the pathogenesis of early brain injury after subarachnoid hemorrhage (SAH). EZH2 (enhancer of zeste homolog 2)-mediated H3K27Me3 (trimethylation of histone 3 lysine 27) has been recognized to play a critical role in multiple inflammatory diseases. However, there is still a lack of evidence to address the effect of EZH2 on the immune response of SAH. Therefore, the aim of this study was to determine the role of EZH2 in SAH-induced neuroinflammation and explore the effect of EZH2 inhibition with its specific inhibitor EPZ6438. METHODS: The endovascular perforation method was performed on rats to induce subarachnoid hemorrhage. EPZ6438, a specific EZH2 inhibitor, was administered intraperitoneally at 1 hour after SAH. SOCS3 (Suppressor of cytokine signaling 3) siRNA and H3K27me3 CRISPR were administered intracerebroventricularly at 48 hours before SAH to explore potential mechanisms. The SAH grade, short-term and long-term neurobehavioral tests, immunofluorescence staining, and western blots were performed after SAH. RESULTS: The expression of EZH2 and H3K27me3 peaked at 24 hours after SAH. In addition, inhibition of EZH2 with EPZ6438 significantly improved neurological deficits both in short-term and long-term outcome studies. Moreover, EPZ6438 treatment significantly decreased the levels of EZH2, H3K27Me3, pathway-related proteins TRAF6 (TNF [tumor necrosis factor] receptor family 6), NF-κB (nuclear factor-κB) p65, proinflammatory cytokines TNF-α, IL (interleukin)-6, IL-1ß, but increased the expression levels of SOCS3 and anti-inflammatory cytokine IL-10. Furthermore, administration of SOCS3 siRNA and H3k27me3-activating CRISPR partly abolished the neuroprotective effect of EPZ6438, which indicated that the neuroprotective effect of EPZ6438 acted, at least partly, through activation of SOCS3. CONCLUSIONS: In summary, the inhibition of EZH2 by EPZ6438 attenuated neuroinflammation via H3K27me3/SOCS3/TRAF6/NF-κB signaling pathway after SAH in rats. By targeting EZH2, this study may provide an innovative method to ameliorate early brain injury after SAH.

NT-4 attenuates neuroinflammation via TrkB/PI3K/FoxO1 pathway after germinal matrix hemorrhage in neonatal rats.

BACKGROUND: Neuroinflammation plays an important role in pathogenesis of germinal matrix hemorrhage (GMH). Neurotrophin-4 (NT-4) is a member of the neurotrophin family and interacts with the tropomyosin receptor kinase B (TrkB). NT-4 has been shown to confer neuroprotective effects following cerebral ischemia. We aimed to investigate the neuroprotective function of NT-4-TrkB signaling, as well as its downstream signaling cascade phosphatidylinositol-3-kinases (PI3K)/protein kinase B (Akt)/forkhead box protein O1 (FoxO1), following GMH in neonatal rats. METHODS: GMH was induced by intraparenchymal injection of bacterial collagenase (0.3 U) in P7 rat pups. A total of 163 pups were used in this study. Recombinant human NT-4 was administered intranasally at 1 h after the collagenase injection. The selective TrkB antagonist ANA-12, selective PI3K inhibitor LY294002, and FoxO1 activating CRISPR were administered intracerebroventricularly at 24 h prior to NT-4 treatment to investigate the underlying mechanism. Short-term and long-term neurobehavioral assessments, immunofluorescence staining, Nissl's staining, and Western blot were performed. RESULTS: Expression of phosphorylated TrkB increased after GMH, reaching the peak level at day 3 after hemorrhage. TrkB receptors were observed on neurons, microglia, and astrocytes. The administration of rh-NT-4 induced phosphorylation of TrkB, expression of PI3K, and phosphorylation of Akt. Meanwhile, it decreased FoxO1 and IL-6 levels. Selective inhibition of TrkB/PI3K/Akt signaling in microglia increased the expression levels of FoxO1 and pro-inflammatory cytokines. FoxO1 activating CRISPR increased the expression of IL-6, suggesting that FoxO1 might be a potential inducer of pro-inflammatory factors. These results suggested that PI3K/Akt/FoxO1 signaling may be the downstream pathway of activation of TrkB. The rat pups treated with rh-NT-4 performed better than vehicle-treated animals in both short-term and long-term behavioral tests. CONCLUSION: These data showed that rh-NT-4 reduced the expression levels of pro-inflammatory cytokines, improved neurological function, attenuated neuroinflammation, and thereby mitigated post-hemorrhagic hydrocephalus after GMH by TrkB/PI3K/Akt/FoxO1 pathway. These results indicated that rh-NT-4 could be a promising therapeutic strategy to ameliorate neuroinflammation and hydrocephalus after GMH or other similar brain injuries.

IRE1α inhibition attenuates neuronal pyroptosis via miR-125/NLRP1 pathway in a neonatal hypoxic-ischemic encephalopathy rat model.

BACKGROUND: Inhibition of inositol-requiring enzyme-1 alpha (IRE1α), one of the sensor signaling proteins associated with endoplasmic reticulum (ER) stress, has been shown to alleviate brain injury and improve neurological behavior in a neonatal hypoxic-ischemic encephalopathy (HIE) rat model. However, there is no information about the role of IRE1α inhibitor as well as its molecular mechanisms in preventing neuronal pyroptosis induced by NLRP1 (NOD-, LRR- and pyrin domain-containing 1) inflammasome. In the present study, we hypothesized that IRE1α can degrade microRNA-125-b-2-3p (miR-125-b-2-3p) and activate NLRP1/caspased-1 pathway, and subsequently promote neuronal pyroptosis in HIE rat model. METHODS: Ten-day old unsexed rat pups were subjected to hypoxia-ischemia (HI) injury, and the inhibitor of IRE1α, STF083010, was administered intranasally at 1 h after HI induction. AntimiR-125 or NLRP1 activation CRISPR was administered by intracerebroventricular (i.c.v) injection at 24 h before HI induction. Immunofluorescence staining, western blot analysis, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), brain infarct volume measurement, neurological function tests, and Fluoro-Jade C staining were performed. RESULTS: Endogenous phosphorylated IRE1α (p-IRE1α), NLRP1, cleaved caspase-1, interleukin-1ß (IL-1ß), and interleukin-18 (IL-18) were increased and miR-125-b-2-3p was decreased in HIE rat model. STF083010 administration significantly upregulated the expression of miR-125-b-2-3p, reduced the infarct volume, improved neurobehavioral outcomes and downregulated the protein expression of NLRP1, cleaved caspase-1, IL-1ß and IL-18. The protective effects of STF083010 were reversed by antimiR-125 or NLRP1 activation CRISPR. CONCLUSIONS: IRE1α inhibitor, STF083010, reduced neuronal pyroptosis at least in part via miR-125/NLRP1/caspase-1 signaling pathway after HI.

DKK3 attenuates JNK and AP-1 induced inflammation via Kremen-1 and DVL-1 in mice following intracerebral hemorrhage.

BACKGROUND: Intracerebral hemorrhage (ICH) is the most devastating stroke subtype, with a poor prognosis and few proven treatments. Neuroinflammation is associated with ICH-induced brain injury and unfavorable outcomes. There is growing evidence that Dickkopf (DKK) 3 plays a key role in the adaptive anti-inflammatory and neuroprotective responses following intracerebral hemorrhage. This study aimed to evaluate the protective effects of DKK3 against brain edema and neuroinflammation in a mice model of ICH. METHODS: Male, adult CD1 mice were subjected to sham or ICH surgery using a collagenase injection model. ICH animals received either recombinant DKK3, Kremen-1 siRNA, or DVL-1 siRNA. The neurobehavioral deficits were evaluated at 24 h, 72 h, and 28 days after ICH induction. Western blot and immunofluorescence were employed to examine the expression and localization of DKK3, Kremen-1, Dishevelled-1 (DVL-1), c-JUN N-terminal kinase (JNK), Activator protein-1 (AP-1), cleaved caspase-1, NF-κB, and IL-1ß in the brain. RESULTS: The expression of endogenous DKK3 and DVL-1 was transiently decreased after ICH compared to that in the sham group. Compared to the mice of ICH, exogenous rDKK3 administration reduced the brain water content and affected the neurological functions in ICH mice. Moreover, DKK3 was colocalized with Kremen-1 in microglia. Using a Kremen-1 or DVL-1 siRNA-induced in vivo knockdown approach, we demonstrated that the effects of DKK3 against ICH were mediated, at least partly, by the Kremen-1 and DVL-1 pathways. CONCLUSIONS: DKK3 improves the neurological outcomes, potentially by decreasing JNK/AP-1-mediated inflammation, thereby ameliorating the short- and long-term sequelae after ICH.

Rh-relaxin-2 attenuates degranulation of mast cells by inhibiting NF-κB through PI3K-AKT/TNFAIP3 pathway in an experimental germinal matrix hemorrhage rat model.

BACKGROUND: Mast cells play an important role in early immune reactions in the brain by degranulation and the consequent inflammatory response. Our aim of the study is to investigate the effects of rh-relaxin-2 on mast cells and the underlying mechanisms in a germinal matrix hemorrhage (GMH) rat model. METHODS: One hundred seventy-three P7 rat pups were subjected to GMH by an intraparenchymal injection of bacterial collagenase. Clodronate liposome was administered through intracerebroventricular (i.c.v.) injections 24 h prior to GMH to inhibit microglia. Rh-relaxin-2 was administered intraperitoneally at 1 h and 13 h after GMH. Small interfering RNA of RXFP1 and PI3K inhibitor LY294002 were given by i.c.v. injection. Post-GMH evaluation included neurobehavioral function, Western blot analysis, immunofluorescence, Nissl staining, and toluidine blue staining. RESULTS: Our results demonstrated that endogenous relaxin-2 was downregulated and that RXFP1 level peaked on the first day after GMH. Administration of rh-relaxin-2 improved neurological functions, attenuated degranulation of mast cells and neuroinflammation, and ameliorated post-hemorrhagic hydrocephalus (PHH) after GMH. These effects were associated with RXFP1 activation, increased expression of PI3K, phosphorylated AKT and TNFAIP3, and decreased levels of phosphorylated NF-κB, tryptase, chymase, IL-6, and TNF-α. However, knockdown of RXFP1 and PI3K inhibition abolished the protective effects of rh-relaxin-2. CONCLUSIONS: Our findings showed that rh-relaxin-2 attenuated degranulation of mast cells and neuroinflammation, improved neurological outcomes, and ameliorated hydrocephalus after GMH through RXFP1/PI3K-AKT/TNFAIP3/NF-κB signaling pathway.